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Numerous asthma and atopy loci have been reported in studies demonstrating associations of the asthma-related phenotypes atopy, elevated IgE levels, and bronchial hyperresponsiveness with alleles of microsatellite markers and single-nucleotide polymorphisms (SNPs) within specific cytokine/chemokine and IgE-regulating genes. Although the studies reporting these observations are compelling, most of them lack statistical power. We assessed the nature, pattern, and frequency of SNPs in 24 candidate genes in Iceland and looked for associations with asthma and atopy. We identified 42 SNPs with an average minor allele frequency of 20.3% (asthma) and 20.7% (control). Twenty SNPs (48%) were within coding sequences and 90% of those led to a predicted change in protein sequence. No differences were detected in the allelic frequencies of SNPs in any of these candidate genes between control subjects and the patients with atopic asthma. Moreover, linkage analysis that included 269 patients with atopic asthma uncovered no evidence of linkage to markers associated with these genes. We conclude that this study has failed to produce evidence in support of the notion that variations within these 24 candidate atopy and asthma genes significantly influence the expression of the atopic asthmatic phenotype or contribute to the susceptibility of atopic asthma.
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Keywords: asthma; atopy; genetic variations; linkage; single-nucleotide polymorphism
Bronchial asthma is a complex genetic disorder with variable phenotype (1, 2). The principal signs and symptoms of asthma, including cough, wheezing, and shortness of breath, are largely attributed to inflamed hyperresponsive airways that characterize the pathobiology of the disease (3, 4). Whereas multiple environmental factors are known to both trigger and/or modulate asthma responses, the genetic components of asthma that underlie the susceptibility to react to "asthma triggers" in the environment remain largely unknown. The use of single-nucleotide polymorphisms (SNPs) has become popular in the analysis of complex genetic disorders. In this context, atopic asthma, one of the leading causes of morbidity in children and young adults, presents a model for complex phenotypes amenable to SNP association analysis (5). The identification of variations in specific genes that are involved in mediating the expression of the atopic asthmatic phenotype could lead the field to a specific molecular pathway providing new drug targets. Both physiologic/pharmacologic and genetic studies in animal models and human populations have identified a number of candidate genes and pathways that regulate IgE levels and airway hyperresponsiveness that represent potential new drug targets (10, 11). However, the significance of SNPs and other genetic variations in these genes to atopic asthma is still unproven.
Collectively, more than 100 linkage and association studies have reported more than 500 atopy and asthma loci throughout the genome. The most commonly used experimental design was to associate one of several asthma-associated phenotypes (atopy, IgE levels, and bronchial hyperresponsiveness [BHR]) to microsatellite markers or SNPs within specific helper T cell type 2 (Th2) cytokine- or IgE-regulating genes (10, 12). However, none of the linkage studies found loci that meet reasonable criteria for significance (15), and only a few of the association studies reported qualify for possible significance. In addition, replication of the latter studies has been difficult, suggesting that either there may be a wide range of variability in allelic frequencies of these genes between different races and populations (2, 10, 15), or that the original loci represent false-positive associations or linkages.
In view of the above-described considerations, we analyzed SNPs in selected promoter and coding regions of 24 candidate asthma and atopy genes in the relatively homogeneous population of Iceland (16). In this regard, it should be noted that asthma is among the most common chronic diseases in Iceland, with an overall prevalence rate of 5% (17) or higher (i.e., 7-10%) as suggested by a survey of Icelandic children (18). Odds ratios of affected offsprings in Iceland are also comparable to those reported in other European countries, or 2.7 (17). In an attempt to increase the sensitivity of our study in determining the roles of these candidate genes in the pathogenesis of the asthma-related phenotypes, a linkage study was performed with microsatellite markers obtained from a genome-wide search (GWS) in the regions of these candidate genes. The candidate genes under study were primarily cytokine genes of the Th1 and Th2 phenotypes and their receptors, growth factors, hormones, and enzymes that have been shown to harbor genetic variants, many of which have been implicated in the pathobiology of asthma (10, 19, 20). SNPs were examined in 94 atopic asthmatic patients and 94 nonatopic nonasthmatic subjects in Iceland. The principal goals of the study were to detect, quantify, and establish potential patterns of sequence variations within selected coding and promoter regions of these candidate atopy and asthma genes that in a significant way associate with atopy and asthma, and to determine whether microsatellite markers could be used to establish linkage of these diseases to these genes.
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METHODS |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Patients
Patients were selected from the private and outpatient clinics of allergists practicing at the Allergy/Immunology and Pulmonary Divisions
of the Vifilstadir University Hospital of Iceland in the years 1977 to
2000. To qualify for the study, the patients had to fulfill either one of
two criteria: a positive skin test to common aeroallergens, and/or carry
a diagnosis of asthma (17). Of 1,982 medical charts reviewed, 1,185 patients were identified who belong to 409 family clusters in which each
patient was related to at least one other patient within four meioses
(e.g., four meiotic events separate a proband from a first cousin) (Figure 1). Eight such family clusters have 10 or more patients, and the largest cluster contains 24 patients. For the SNP studies, 94 patients
with atopy and asthma were randomly selected, 1 from each of 94 of
the 409 available family clusters; for the linkage studies, an additional
175 patients were selected from these same 94 family clusters to assure
that each of the 269 patients was related to at least one other atopic/
asthmatic patient within four meioses. Ages ranged from 12 to 59 yr
(mean, 38 yr) and 59.9% were females. Information regarding age at
diagnosis, medications, hospital admissions, and family history of
atopy and asthma were collected. The atopic and asthma phenotypes
were characterized on the basis of medical history, physical examination, skin tests to 12 aeroallergens (including birch, grass, Rumex acetocella, cat, dog, horse, Cladosporium, Mucor, Alternaria, Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Lepidoglyphus
destructor), total IgE levels, pulmonary function tests (PFTs), and, unless baseline FEV1 was 
70% predicted, a methacholine (MCh) challenge test. The phenotype assessments, PFTs, and methacholine tests were performed according to American Thoracic Society (ATS) guidelines (21, 22). Patients were diagnosed as being atopic if their skin
prick test reaction was 
3 mm or 50% of the histamine positive
control response. The participation rate of the patients in the study exceeded 90%. All patients signed an informed consent form, donated
blood samples, and completed a questionnaire and all tests necessary
for proper phenotyping. The study was approved by the Icelandic Data
Protection Commission and the National Bioethics Committee. Personal information about the patients and their family members were
subsequently encrypted by the Data Protection Commission of Iceland
(23). All blood and DNA samples were also coded in the same way.
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Control Subjects
Ninty-four unrelated subjects without history of asthma and atopy, matched for age and sex with the 94 patients with atopic asthma, were randomly selected from more than 300 healthy subjects who donated blood to serve as control subjects for the association studies. These individuals filled out a questionnaire that included information about diseases and medication use. These individuals did not undergo a physical examination and no tests were performed, either.
Genealogy Database and Cluster Function
deCODE has built a computerized genealogy database with 630,000 names that includes all 285,000 living Icelanders and most of their ancestors (24). The database has a connectivity rate of 95% in the 20th century and 86% in the 19th century. Its maternal connections are 99.3% accurate as measured by examining the mitochondrial sequences of maternally linked individuals (16). Paternal accuracy exceeds 98.5%. Each record in the database consists of a personal identifier, identifier to parents, sex, phenotype, and approximate dates of birth and death. All personal identifiers in the genealogy database are reversibly coded by the Data Protection Commission outside our laboratory. Algorithms were developed that find all ancestors in the database who are related to each member of the input list within a given number of generations. Using these groups, the cluster function searches for ancestors who are common to any two or more members of the input lists. This information was then used to assure that the patients who participated in the association studies were unrelated. In contrast, the patients who participated in the linkage study were all connected to at least one other affected patient within six meioses. The Data Protection Commission of Iceland coded lists of patients participating in the study in the same manner as the genealogy database (23).
Candidate Asthma and Atopy Gene Selection
We identified "candidate atopy and asthma genes" on the basis of their reported involvement in atopy and asthma and their polymorphism. This resulted in a collection of more than 50 candidate genes. We selected a subset of 25 genes for the primary polymorphism study because of their strengths as candidate genes based on previous studies as well as the availability of genomic sequence, obtained from GenBank and/or publications (See Table 1). Analysis of 24 genes was successfully completed. One gene (the 5-lipoxygenase [5-LO] gene) sequenced poorly and was excluded from the analysis because of incomplete data sequences.
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SNP Detection
For the SNP detection experiment, we used gel-based nucleotide sequencing of relevant polymerase chain reaction (PCR) products from individual samples, using product-specific primers for the targeted promoter and exonic gene regions (http://inotes.decode.is/asthma.nsf ). The DNA was sequenced from the 94 subjects with atopic asthma and compared with a control group that was also randomly selected from age- and sex-matched, nonatopic, nonasthmatic individuals who were not related to each other within six meioses. All SNPs were classified as either homozygous or heterozygous for an alternative (relative to the reference) base. Genomic DNA was isolated from the peripheral blood cells of subjects, using the sucrose and phenol-chloroform extraction method (25). PCRs were 20 µl in volume and used 20 ng of template genomic DNA, 0.25 U of AmpliTaq Gold, 2 pmol of each primer, 0.2 mM deoxynucleotides, and 2.5 mM MgCl2. The products were electrophoresed and detected on an Applied Biosystems (Foster City, CA) model 377 sequencer. After gel tracking the sequences were analyzed with Seqman II DNAStar (Madison, WI) software. We report only genetic variants in this study that are "confirmed" as determined in separate analysis from both forward and reverse PCR products by two skilled scientists, thereby minimizing the error rates.
Genotyping
Apart from the 94 patients who were analyzed for SNPs, DNA was also extracted for genotyping from the peripheral blood of an additional 175 of their affected relatives and 230 of their unaffected relatives, the latter including both parents in 60% of cases, one parent together with one or two siblings in 30% of cases, and two or three siblings if neither parent was available in 10% of cases. All DNA samples were genotyped with specific fluorescently labeled primers with an average spacing of 3-4 cM in the chromosomal regions of these candidate atopy and asthma genes (26, 27). PCRs were set up, run, and pooled on Perkin-Elmer/Applied Biosystems 877 integrated catalyst thermocyclers as previously described (25). The PCR conditions used were 95° C for 10 min to activate the AmpliTaq Gold, and then 34 cycles of denaturation at 94° C for 15 s, annealing at 55° C for 30 s, and elongation at 72° C for 1 min. The pooled products were supplemented with the internal size standards and were electrophoresed and detected on an Applied Biosystems model 377 sequencer, using GeneScan version 3.0 peak calling software. The genotypes were defined and edited in the Applied Biosystems Genotyper version 2.0 program. The marker orders and genetic distances used were obtained from both publicly available genetic maps and genetic maps constructed at deCODE Genetics (24). Two separate reference samples of northern European descent from the CEPH (Centre d'Etude du Polymorphisme Humain; http://www.cephb.fr/ ) collection (1331-01 and 1331-02; Coriell Cell Repositories, Camden, NJ) were used.
Statistical Analysis
Statistical analysis of allelic associations of all SNPs within candidate
asthma genes was performed by Student t test and 
2 test. Odds ratios and
95% confidence intervals for all 42 SNPs are included. All linkage results
reported were produced by the Allegro program (28), which includes enhanced statistical analytical features of the Genehunter-Plus program (29,
30). The Allegro program implements a nonparametric method of linkage
analysis and produces nonparametric linkage (NPL) scores, which indicate the amount of excess identity by descent (IBD) sharing among related affecteds as measured by a chosen scoring function (28). The scoring
function used for this manuscript was Spairs, which has been found to be
powerful for a wide range of inheritance models with or without locus heterogeneity (30, 31). LOD (log10 of the odds) scores for microsatellite
markers in the regions of the candidate atopy and asthma genes (i.e., genes
that were evaluated for SNPs) are reported for the purpose of comparing
variation in SNP frequency in the context of the regional LOD scores.
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RESULTS |
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DISCUSSION
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Characteristics of Patients
Family clusters were built by comparing the encrypted patient lists with the genealogy database, encrypted the same way (see METHODS). This revealed 409 family clusters with 1,185 patients, two-thirds of whom have atopy and asthma, with the balance evenly split between atopy alone and asthma without atopy. Two hundred and sixty-nine patients with atopic asthma participated in this study. Of the 94 patients who were analyzed for SNPs, more than two-thirds had moderate to severe asthma, and their mean maintenance dose of inhaled corticosteroids was 600 µg/d (range, 200-1,600 µg/d). The additional 175 patients who were genotyped for linkage analysis were selected from the same 94 family clusters and were related to 1 or more of the above-described 94 patients within four meioses. The top row in Table 2 shows the geometric mean IgE values, the mean percent predicted values of FEV1 (using the lowest values available for a given patient), and the results of the methacholine challenge tests for the 94 patients with atopic asthma who were analyzed for SNPs associations (e.g., Group 1). The bottom row of Table 2 shows comparable measurements for all 269 patients who were used for linkage analysis (e.g., Group 2), including percent predicted FEV1 values that all patients performed when entering the study. Patients skin test results to the 12 most common aeroallergens in Iceland are shown in Table 3.
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Variations in Candidate Asthma Genes Determined by Sequencing
We examined approximately 11 kb of genomic sequence comprising selected promoter and exon regions in 24 candidate genes in 94 Icelandic patients with atopic asthma and 94 Icelandic subjects without atopy or asthma, or approximately 4.1 Mb of DNA. Data were obtained by sequencing of PCR products from genomic DNA, using specific primers. We identified 42 candidate SNPs in the promoter regions and selective exon sequences of these 24 candidate genes (Table 4) (19, 32). Eighteen (43%) of these SNPs had alleles that encoded two amino acids. Apart from SNPs in eight genes belonging to the gene cluster on chromosome 5q31-33, the other genes examined were located on 11 different chromosomes across the genome. Overall, the frequencies of the major alleles of the sequence-altering SNPs were lower in the atopy and asthma population in Iceland compared with those reported in other atopy and asthma populations in Western societies (see references listed in Table 4). In contrast, the average minor allele frequency of 24 of the most common SNPs for which reference data were available was similar in Iceland at 22.1% compared with 23.2% in other populations. When taken together with reports obtained from analysis of mitochondrial haplotypes (16), the latter data support the notion that the Icelandic population is relatively homogeneous (16). However, in contrast to other reports (10, 19, 20), we detected no differences in the allelic frequencies of SNPs in any of these 24 candidate atopy and asthma genes between control subjects and the atopic asthmatic patients in Iceland (i.e., 20.7 versus 20.3% overall, respectively).
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Patterns of SNPs in Candidate Genes for Asthma and Atopy
We also looked for any differences in the patterns of allelic
distributions between multiple SNPs in these candidate genes. Each SNP was correlated with all possible combinations of
two, three, or four other SNPs, both within and between different genes, in patients and control subjects (i.e., multilocus
SNP analysis). Significant linkage disequilibrium was observed between several of the SNPs, particularly between
SNPs that were located within the same gene (p values as low
as 10
38). However, there were no differences observed in the
patterns of SNP correlations between control subjects and the
patients with atopic asthma (data not shown).
Linkage Results
To search further for roles of these SNPs or other potential genetic variability within these candidate atopy and asthma genes in the expression of the atopic asthmatic phenotype, a linkage analysis was carried out with microsatellite markers in the regions of the above-described 24 candidate atopy and asthma genes. The 94 atopic asthmatic families that were examined for linkage included the 94 patients who took part in the association studies, together with 175 of their affected relatives (e.g., total of 269 patients with atopic asthma) and 230 of their unaffected relatives. As shown in Figure 2, there was no evidence of linkage to microsatellite markers in or close to any of these 24 candidate atopy and asthma susceptibility genes.
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Cumulative evidence suggests that the expression of the atopic asthmatic phenotype is due to complex interactions among multiple "unknown" atopy and asthma susceptibility genes and between them and the environment (10, 12, 75). Whereas several linkage and association studies have suggested the presence of various candidate atopy and asthma genes, it has proven difficult to detect consistent abnormalities in these genes that may have disease-modifying effects (10, 12, 40). Because sequence variations in the coding regions of human genes are largely confined to SNPs, screening candidate genes for SNPs has been a popular method in the hunt for disease-modifying genes for complex genetic disorders, and is potentially useful in tests of association with common traits (7, 8, 28, 76, 77). Several SNPs have been detected within candidates for atopy and asthma genes that may confer alterations in the expression of these genes and the function of their products. An example of such alterations is the R576 mutation in the interleukin 4 (IL-4) receptor gene on the short arm of chromosome 16 (72), and the variations at positions 16 and 27 in the ADRB2 gene on the long arm of chromosome 5 (37). However, there is a wide range in the prevalence of these genetic variations between ethnic groups and populations and their roles as disease-modifying genes remain uncertain (5, 11).
This study examined the genomic sequences in selected
promoter and exon regions of 24 candidate genes in 94 Icelandic patients with atopic asthma and 94 Icelandic subjects without atopy and asthma, and searched for allelic associations between markers and atopic asthma. Furthermore, we searched
for linkages between markers in the regions where the 24 genes are and atopy and asthma. The regions examined constituted approximately 4.1 Mb of DNA and included 42 SNP
variants in these genes. As shown in Table 1, the targeted
genes that were sequenced included the cytokine gene cluster
on chromosome 5q31-33, the IL-4 receptor on chromosome 16p12 (19), the Fc
RI receptor gene on chromosome 11q13
(37), as well as other genes encoding cytokine/chemokines and
adhesion molecules and genes encoding various metabolizing
enzymes that have been implicated in the pathogenesis of
asthma (see references in Table 4). The allelic frequencies of
each polymorphism, which included primarily SNPs that alternate amino acids and others in critical regulatory regions, are
shown in Table 4. The overall frequencies of the major protein-altering SNPs examined in the Icelandic population are
similar to those reported in other populations of European descent (16, 79, 80), lending further support to the concept that
the gene pool and the genetic variability of the Icelandic population are broad and that genetic research in this population
is likely to yield results that are relevant to more heterogeneous populations. As demonstrated in Table 4, there were no
differences in the allelic frequencies of any of the SNPs detected, nor was there any difference in the patterns of combinations of SNPs in these 24 candidate atopy and asthma genes
between control subjects and the patients with atopic asthma. In this regard, it should be noted that we were rigorous in the selection of the patient phenotypes (see METHODS and Table
2). We cannot exclude that a complex epistasis/interaction between the various SNPs that has eluded our analysis contributes to the atopic asthmatic phenotype. However, our linkage
studies conducted with 269 atopic, asthmatic subjects and 230 of their unaffected immediate relatives demonstrated no evidence of linkage to microsatellite markers that are associated
with each of these 24 candidate atopy and asthma genes, suggesting that SNPs or other genetic variations in these candidate atopy and asthma genes are unlikely to have large disease-modifying effects in the Icelandic population.
To our knowledge, this is the first study to simultaneously report on allelic frequency and pattern of SNPs together with linkage results for multiple candidate atopy and asthma genes. Given that the minor allele frequencies for several of these SNPs is reportedly higher in atopy and asthma populations of other Western societies compared with controls, we would have expected to see a trend toward increased minor allele frequency in at least a few of these SNPs in the Icelandic asthma and atopy population if these SNPs were important in determining the genetic susceptibility to atopy and asthma. However, to our surprise, we detected no differences in the allelic frequencies of SNPs in any of these 24 candidate atopy and asthma genes between control subjects and patients in Iceland (see Table 4). To address the issue of study power we calculated 95% confidence intervals (CI) and examined odds ratios (OR) as shown in Table 4. We also calculated the power of the study to identify alleles that would carry a relative risk of 2 or more. Overall, this study has 80% power of determining whether any of the alleles examined carries a risk of 2 or more (p < 0.05). Moreover, because none of the p values reported in Table 4 are close to significant for any of the associations examined for these 42 SNPs in our cohort of 94 patients and 94 controls, together with low OR values (highest, 1.39) and a relatively narrow CI range, we believe it is unlikely that we are missing a significant association (i.e., with p < 0.05) with any of these SNPs. Indeed, several of the alleles that have been associated with atopy and/or asthma in other populations showed opposite association with the disease in Iceland (38, 49, 50, 65, 72). Thus, it would be unlikely that a comparable significance would be uncovered in the Icelandic population by increasing the number of patients in the study.
Taken together, these data support the concept that the risk that these candidate asthma and atopy genes may confer is small, if any, and that a relative risk of 2 or higher can be excluded. Thus, the additional power that would be gained from increasing the study cohort would not be likely to change these results or lead to significantly increased disease-associated allele prevalence for these SNPs in patients relative to control subjects. In evaluating the relevance of these results, it is noteworthy that it has been generally accepted that founder populations may be advantageous when selecting control subjects to cases, given their relatively homogeneous nature (81). The fact that we were unable to replicate the results of many of the published studies by our large-scale sequencing together with linkage analysis to these 24 genes in Iceland, suggests that some of the original loci reported may represent false-positive associations or linkages. Although numerous reasons may account for these differences, the latter may be attributed to reports on too few patients and a lack of statistical power, the use of mixed populations in regard to ethnicity and/or sex, laboratory error in genotype detection, or study recruitment bias together with inherited population variability to name a few.
We conclude that this study has failed to produce evidence in support of the notion that SNPs in the 24 candidate asthma and atopy genes examined significantly influence the expression of the atopic asthmatic phenotype or contribute to the susceptibility of atopic asthma.
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Footnotes |
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Correspondence and requests for reprints should be addressed to H. Hakonarson or K. Stefansson, deCODE Genetics, Inc., Lynghals 1, 110 Reykjavik, Iceland. E-mail: hakonh{at}decode.is (H.Hakonarson); kstefans{at}decode.is (K. Stefansson).
(Received in original form January 22, 2001 and accepted in revised form September 17, 2001).
Dr. Hakonarson is supported in part by RO1 grant HL59906.
Acknowledgments:
The authors thank the Icelandic patients, their family
members, and the individuals who served as control subjects for their generous support for this work. They also thank their nurses; study coordinator;
and Genealogy, Bioinformatics, DNA Isolation, GWS, and Sequencing Core
staff for invaluable technical expertise and assistance with the research. The
authors thank the Ursinus College student visitors, E. Valiant, C. Carpenter,
and R. Whelan and their mentor, J. Noveral, for valuable contributions, Andrei Manolescu and John Barnard for assisting with the statistics, and Mark
Gurney for reviewing the manuscript.
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References |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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